Protein Engineering Produces a Molecular
'Switch'

Technique could lead to new drug delivery systems,
biological warfare sensors

By Phil SneidermanHomewood

Using a lab technique called domain insertion, Johns
Hopkins researchers have joined two proteins in a way that
creates a molecular "switch." The result, the researchers
say, is a microscopic protein partnership in which one
member controls the activity of the other. Similarly
coupled proteins may someday be used to produce specialized
molecules that deliver lethal drugs only to cancerous
cells. They also might be used to set off a warning signal
when biological warfare agents are present.

The technique used to produce this molecular switch
was reported March 27 in New Orleans at the 225th national
meeting of the American Chemical Society.

"We've taken two proteins that normally have nothing
to do with one another, spliced them together genetically
and created a fusion protein in which the two components
now 'talk' to one another," said Marc Ostermeier, assistant
professor in the
Department of Chemical and Biomolecular Engineering in
the Whiting School. "More important, we've shown that one
of these partners is able to modulate or control the
activity of the other. This could lead to very exciting
practical applications in medical treatment and
bio-sensing."

In this Johns Hopkins engineering lab, Gurkan Guntas and
Marc Ostermeier used a technique called domain insertion to
join two proteins and create a molecular 'switch.'

To prove the production of a molecular switch is
possible, Ostermeier, assisted by doctoral student Gurkan
Guntas, started with two proteins that typically do not
interact: beta-lactamase and the maltose binding protein
found in a harmless form of E. coli bacteria. Each protein
has a distinct activity that makes it easy to monitor.
Beta-lactamase is an enzyme that can disable and degrade
penicillinlike antibiotics. Maltose binding protein binds
to a type of sugar called maltose that the E. coli cells
can use as food.

Using a technique called domain insertion, the Johns
Hopkins researchers placed beta-lactamase genes inside
genes for maltose binding protein. To do this, they snipped
the maltose binding genes, using enzymes that act like
molecular scissors to cut the genes as though they were
tiny strips of paper. A second enzyme was used to re-attach
these severed strips to each side of a beta-lactamase gene,
producing a single gene strip measuring approximately the
combined length of the original pieces. This random
cut-and-paste process took place within a test tube and
created hundreds of thousands of combined genes. Because
the pieces were cut and reassembled at different locations
along the maltose binding gene, the combined genes produced
new proteins with different characteristics.

Ostermeier believed a very small number of these new
fusion proteins might possess the molecular switch behavior
he was looking for. To find them, he and Guntas took a cue
from the process of evolution, or "survival of the
fittest." By looking for the E. coli that thrived in
maltose, they could isolate only the ones in which the
maltose binding partner was still active (in other words,
it still bound itself to maltose). By then mixing them with
an antibiotic, the researchers could find the ones in which
the beta-lactamase remained active and capable of reacting
against the antibiotic. Through such survival tests, the
researchers ultimately were able to find two fusion
proteins in which not only were both proteins still active
but in which the presence of maltose actually caused the
beta-lactamase partner to step up its attack on an
antibiotic.

"In other words," Ostermeier said, "one part of this
coupled protein sent a signal, telling the other part to
change its behavior. This is the first clear demonstration
that you can apply the domain insertion technique to
control the activity of an enzyme. If we can replicate this
with other proteins, we can create biological agents that
don't exist in nature but can be very useful in important
applications."

For example, Ostermeier said, one part of a fusion
protein might react only to cancer cells, signaling its
partner to release a toxin to kill the diseased tissue.
Healthy cells, however, would not set off the switch and
would thus be left unharmed. Ostermeier also suggested that
one part of a fusion protein might react to the presence of
a biological warfare agent, signaling its partner to set
off a bright green fluorescent glow that could alert
soldiers and others to the danger.

Johns Hopkins has applied for U.S. and international
patents related to Ostermeier's molecular switch technology
and the techniques used to produce them. Ostermeier's
research has been funded by grants from the American Cancer
Society and the Maryland Cigarette Restitution Fund.